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NEDD8 links cullin-RING ubiquitin ligase function to the p97 pathway

Nature Structural & Molecular Biology volume 19, pages 511–516 (2012)Cite this article

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Abstract

The AAA+ ATPase p97 and its UBA-UBX cofactors are thought to extract ubiquitinated proteins from membranes or protein complexes as a prelude to their degradation. However, for many cofactors ubiquitinated targets have not yet been identified, leaving their biological function unclear. Previous analysis has linked the p97 pathway to cullin-RING ubiquitin ligases (CRLs); here we demonstrate that the human p97 cofactor UBXD7 mediates the p97-CRL interaction through its conserved ubiquitin-interacting motif (UIM). UBXD7 and its yeast ortholog, Ubx5, associate only with the active, NEDD8- or Rub1-modified form of cullins. Disruption of the Ubx5 UIM results in a loss of CRL binding and consequently impedes degradation of a Cul3 substrate. These results uncover an unexpected and conserved role for NEDD8 in linking CRL ubiquitin ligase function to the p97 pathway.

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Figure 1: UBXD7 associates with all cullins except CUL5.
Figure 2: The UBXD7-CRL interaction is neddylation dependent.
Figure 3: UBXD7 directly interacts with neddylated CRLs via its UIM.
Figure 4: The UIM of UBXD7 binds conjugated NEDD8 on CUL2.
Figure 5: The UIM in yeast Ubx5 promotes UV-dependent degradation of Rpb1.

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References

  1. Ye, Y. Diverse functions with a common regulator: ubiquitin takes command of an AAA ATPase. J. Struct. Biol. 156, 29–40 (2006).

    Article  CAS  Google Scholar 

  2. Alexandru, G. et al. UBXD7 binds multiple ubiquitin ligases and implicates p97 in HIF1alpha turnover. Cell 134, 804–816 (2008).

    Article  CAS  Google Scholar 

  3. Deshaies, R.J. & Joazeiro, C.A. RING domain E3 ubiquitin ligases. Annu. Rev. Biochem. 78, 399–434 (2009).

    Article  CAS  Google Scholar 

  4. Jin, J. et al. Systematic analysis and nomenclature of mammalian F-box proteins. Genes Dev. 18, 2573–2580 (2004).

    Article  CAS  Google Scholar 

  5. Lee, J.E. et al. The steady-state repertoire of human SCF ubiquitin ligase complexes does not require ongoing Nedd8 conjugation. Mol. Cell. Proteomics 10, M110.006460 (2011).

    Article  Google Scholar 

  6. Mahrour, N. et al. Characterization of Cullin-box sequences that direct recruitment of Cul2-Rbx1 and Cul5-Rbx2 modules to Elongin BC-based ubiquitin ligases. J. Biol. Chem. 283, 8005–8013 (2008).

    Article  CAS  Google Scholar 

  7. Saha, A. & Deshaies, R.J. Multimodal activation of the ubiquitin ligase SCF by Nedd8 conjugation. Mol. Cell 32, 21–31 (2008).

    Article  CAS  Google Scholar 

  8. Duda, D.M. et al. Structural insights into NEDD8 activation of cullin-RING ligases: conformational control of conjugation. Cell 134, 995–1006 (2008).

    Article  CAS  Google Scholar 

  9. Bosu, D.R. & Kipreos, E.T. Cullin-RING ubiquitin ligases: global regulation and activation cycles. Cell Div. 3, 7 (2008).

    Article  Google Scholar 

  10. Soucy, T.A. et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–736 (2009).

    Article  CAS  Google Scholar 

  11. Uchiyama, K. et al. VCIP135, a novel essential factor for p97/p47-mediated membrane fusion, is required for Golgi and ER assembly in vivo. J. Cell Biol. 159, 855–866 (2002).

    Article  CAS  Google Scholar 

  12. Bennett, E.J., Rush, J., Gygi, S.P. & Harper, J.W. Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell 143, 951–965 (2010).

    Article  CAS  Google Scholar 

  13. Hofmann, K. & Falquet, L. A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems. Trends Biochem. Sci. 26, 347–350 (2001).

    Article  CAS  Google Scholar 

  14. Young, P., Deveraux, Q., Beal, R.E., Pickart, C.M. & Rechsteiner, M. Characterization of two polyubiquitin binding sites in the 26 S protease subunit 5a. J. Biol. Chem. 273, 5461–5467 (1998).

    Article  CAS  Google Scholar 

  15. Fisher, R.D. et al. Structure and ubiquitin binding of the ubiquitin-interacting motif. J. Biol. Chem. 278, 28976–28984 (2003).

    Article  CAS  Google Scholar 

  16. Whitby, F.G., Xia, G., Pickart, C.M. & Hill, C.P. Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes. J. Biol. Chem. 273, 34983–34991 (1998).

    Article  CAS  Google Scholar 

  17. Hirano, S. et al. Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting. Nat. Struct. Mol. Biol. 13, 272–277 (2006).

    Article  CAS  Google Scholar 

  18. Gray, J.J. et al. Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations. J. Mol. Biol. 331, 281–299 (2003).

    Article  CAS  Google Scholar 

  19. Yamoah, K. et al. Autoinhibitory regulation of SCF-mediated ubiquitination by human cullin 1's C-terminal tail. Proc. Natl. Acad. Sci. USA 105, 12230–12235 (2008).

    Article  CAS  Google Scholar 

  20. Verma, R., Oania, R., Fang, R., Smith, G.T. & Deshaies, R.J. Cdc48/p97 mediates UV-dependent turnover of RNA Pol II. Mol. Cell 41, 82–92 (2011).

    Article  CAS  Google Scholar 

  21. Schuberth, C. & Buchberger, A. UBX domain proteins: major regulators of the AAA ATPase Cdc48/p97. Cell. Mol. Life Sci. 65, 2360–2371 (2008).

    Article  CAS  Google Scholar 

  22. Ribar, B., Prakash, L. & Prakash, S. ELA1 and CUL3 are required along with ELC1 for RNA polymerase II polyubiquitylation and degradation in DNA-damaged yeast cells. Mol. Cell. Biol. 27, 3211–3216 (2007).

    Article  CAS  Google Scholar 

  23. Rabut, G. et al. The TFIIH subunit Tfb3 regulates cullin neddylation. Mol. Cell 43, 488–495 (2011).

    Article  CAS  Google Scholar 

  24. Hicke, L., Schubert, H.L. & Hill, C.P. Ubiquitin-binding domains. Nat. Rev. Mol. Cell Biol. 6, 610–621 (2005).

    Article  CAS  Google Scholar 

  25. Kleiger, G., Saha, A., Lewis, S., Kuhlman, B. & Deshaies, R.J. Rapid E2–E3 assembly and disassembly enable processive ubiquitylation of cullin-RING ubiquitin ligase substrates. Cell 139, 957–968 (2009).

    Article  CAS  Google Scholar 

  26. Matsuoka, S. et al. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316, 1160–1166 (2007).

    Article  CAS  Google Scholar 

  27. Stokes, M.P. et al. Profiling of UV-induced ATM/ATR signaling pathways. Proc. Natl. Acad. Sci. USA 104, 19855–19860 (2007).

    Article  CAS  Google Scholar 

  28. Higa, L.A. & Zhang, H. Stealing the spotlight: CUL4–DDB1 ubiquitin ligase docks WD40-repeat proteins to destroy. Cell Div. 2, 5 (2007).

    Article  Google Scholar 

  29. Hua, Z. & Vierstra, R.D. The cullin-RING ubiquitin-protein ligases. Annu. Rev. Plant Biol. 62, 299–334 (2011).

    Article  CAS  Google Scholar 

  30. Chew, E.H. & Hagen, T. Substrate-mediated regulation of cullin neddylation. J. Biol. Chem. 282, 17032–17040 (2007).

    Article  CAS  Google Scholar 

  31. Huang, D.T. & Schulman, B.A. Expression, purification, and characterization of the E1 for human NEDD8, the heterodimeric APPBP1-UBA3 complex. Methods Enzymol. 398, 9–20 (2005).

    Article  CAS  Google Scholar 

  32. Li, T., Pavletich, N.P., Schulman, B.A. & Zheng, N. High-level expression and purification of recombinant SCF ubiquitin ligases. Methods Enzymol. 398, 125–142 (2005).

    Article  CAS  Google Scholar 

  33. Petroski, M.D. & Deshaies, R.J. In vitro reconstitution of SCF substrate ubiquitination with purified proteins. Methods Enzymol. 398, 143–158 (2005).

    Article  CAS  Google Scholar 

  34. Pierce, N.W., Kleiger, G., Shan, S.O. & Deshaies, R.J. Detection of sequential polyubiquitylation on a millisecond timescale. Nature 462, 615–619 (2009).

    Article  CAS  Google Scholar 

  35. Chen, Z. & Pickart, C.M. A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin. J. Biol. Chem. 265, 21835–21842 (1990).

    CAS  Google Scholar 

  36. Longtine, M.S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Rome for providing Lys48-linked polyubiquitin chains, N. Pierce for purified SCFCdc4, T. Hagen (National University of Singapore) and E. Emberley for cullin expression constructs, and D. Duda and B. Schulman (St. Jude Children's Research Hospital) for providing purified recombinant CUL2, CUL3, CUL4a, CUL1ΔWHB and NCE2 proteins. We are grateful to S. Lewis for help with the Rosetta Dock server, Millennium Pharmaceuticals for MLN4924 and NEDD8 antibody, and the members of the Deshaies laboratory for helpful discussion during the course of this work. This work was supported by the Howard Hughes Medical Institute (HHMI) and a National Institute of Health Ruth Kirschstein Postdoctoral Fellowship (F32 GM088975; W.d.B.). R.J.D. is an HHMI Investigator.

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Authors and Affiliations

  1. Division of Biology, California Institute of Technology, Pasadena, California, USA

    Willem den Besten, Rati Verma, Gary Kleiger, Robert S Oania & Raymond J Deshaies

  2. Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, USA

    Rati Verma, Gary Kleiger, Robert S Oania & Raymond J Deshaies

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  1. Willem den Besten
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Contributions

W.d.B. and R.J.D. conceived and designed the experiments; W.d.B. performed most of the experiments, except that G.K. performed the structural modeling in Figure 3a, and R.V. and R.S.O. made the yeast strains and carried out the Rbp1 turnover studies in Figure 5b and c; W.d.B. and R.J.D. wrote the manuscript with editorial input from the other authors.

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Correspondence to Raymond J Deshaies.

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R.J.D. is a founder and shareholder of Cleave Biosciences.

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den Besten, W., Verma, R., Kleiger, G. et al. NEDD8 links cullin-RING ubiquitin ligase function to the p97 pathway. Nat Struct Mol Biol 19, 511–516 (2012). https://doi.org/10.1038/nsmb.2269

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